Method for determination of likelihood of occurrence of preterm labor in pregnant females

ABSTRACT

The presence of 3-chlorotyrosine in vaginal secretions in a predictor of the likelihood preterm premature rupture of the fetal membranes, i.e., the chorioamnion and/or the occurrence of the risk of preterm labor. 3-chlorotyrosine is a marker for the excessive production of hypochlorous acid which causes focal areas of increased collagen destruction in the chorioamnion, preterm premature rupture of membranes, or increased production of postaglandin and preterm labor. The inventive method comprises determining the likelihood of the occurrence of preterm premature rupture of membranes or increased production of postaglandin and preterm labor in a pregnant female by obtaining a sample of the females vaginal secretions, and analyzing the secretions for the presence and amount of 3-chlorotyrosine in the sample. Various antibody-based tests can be used to measure 3-chlorotyrosine, and a number of neoantigens, useful in raising antibodies to 3-chlorotyrosine, are disclosed.

BACKGROUND OF THE INVENTION

1. Area of the Art

This invention relates to the field of accurate identification ofpregnant women who are at risk for the occurrence of preterm laborgiving birth to extremely low weight babies with low likelihood chanceof survival.

2. Description of the Prior Art

Preterm labor (PTL) leading to premature birth is a leading cause ofboth infant mortality and long-term disabilities. While there have beensome treatments proposed (see U.S. published Patent Application2001/0031731 to Buhimschi et al., the contents of which are incorporatedherein by reference), there is a serious problem in deciding when suchtreatments should be instituted and whether the treatment is at alleffective. Especially as it is not clear that all of the possible PTLtreatments are without potential harm. Thus, one must be reluctant toinstitute a treatment where the need for treatment is uncertain. Onepredictor for PTL is previous instances of PTL in the same individual.However, that is somewhat like closing the barn door after the escape.It would be much better (and cost effective) to detect actual markers ofPTL.

Currently, fetal fibronectin (fFN) in vaginal secretions is theprincipal FDA approved biomarker for PTL. Fetal fibronectin is anadhesion molecule that binds the amnion layer in the fetal membrane tothe chorion layer beneath it. Typically, fFN can be detected in vaginalsecretions early in pregnancy (<20 weeks' gestation), reflecting thefact that the chorion and amnion do not fuse until 20 weeks. From 20 to34 weeks' gestation, no fFN is detected in vaginal secretions. After 34weeks' gestation, fFN begins to appear, presumably reflecting someseparation of chorion and amnion as the collagen matrix remodels inpreparation for labor at term. During this time, MMP-9, the enzyme thatdegrades collagen IV in basement membranes, increases ultimately toweaken the fetal membranes for the birth process.

Fetal fibronectin, when detected in <34 weeks' gestations, has beeninterpreted to indicate an increased risk of PTL. Presumably theagitation that occurs as premature labor begins produces shearing forcesthat lead to release of fFN into the vaginal secretions. Depending onthe study, the presence of fFN in vaginal fluid is associated with a 20to 50% chance of PTL. On the other hand, absence of fFN is associatedwith a 99% chance of not going into labor prematurely. This test, then,is much more useful for its negative predictive value than its positivepredictive value.

Analysis of the chorioamnion shows that its strength is the result ofcollagen. Collagen is produced by fibroblasts in the amnion and chorion.Results of culturing amnion epithelial cells indicate that there isactive biosynthesis and secretion of collagenous matrix up to term (1).Five types of collagen have been identified, types I, III, IV, V and VI(out of 12 known collagen types). Types I, III, V and VI are organizedin triple helices. The strength for these collagens is derived fromtheir helix configurations and hydroxyproline and hydroxylysine bridgesacross the helix. Type IV collagen is different from the other fourtypes in that it forms a mesh as part of the basement membrane (21).

Matrix metalloproteinase I (MMP-1) degrades types I, II, and IIIcollagens; MMP-2 and MMP-9 degrade type IV collagen (31). MMP-3, 7, 10,and 11 have broad substrate specificity. Release of matrixmetalloproteinase is controlled by tissue inhibitors ofmetalloproteinases or TIMPS that bind with metalloproteinases andprevent them from degrading the collagen (21).

Tensile strength and thickness measurements of the chorioamnion indicatethat the membranes are thinner at the rupture site than over theplacenta (Artel 1976 supra). Moreover, preterm membranes in general arestronger than term PROM or spontaneous rupture of membrane (SROM)membranes suggesting that PPROM represents a local defect (15).

Metalloproteinase 9 (which degrades collagen IV) has been shown to beupregulated naturally at term in association with a reduction inmembrane tensile strength (27).

Infection, or culturing membranes with Escherichia colilipopolysaccharide or group A streptococcus polysaccharide also has beenshown to increase MMP 9 (7). Increased MMP-9 also was noted whenchorioamnion segments were cultured with the ROS, superoxide (5).

It would appear that Inflammation, therefore, plays a role in PPROM.Increased MMP-9 is found in amniotic fluid of term PROM patients andthose in labor but not in patients undergoing elective cesarean section(28). Likewise, MMP-3 in amniotic fluid is increased three-fold in PPROMpatients over term controls (8) Stromelysins in placental membranes andamniotic fluid with premature rupture of membranes. Others have reportedthat MMP-1 is increased in amniotic fluid of PPROM patients and is evenhigher if bacteria are present in the fluid (17).

SUMMARY OF THE INVENTION

We have discovered an improved method for predicting the likelihood ofthe occurrence of preterm premature rupture of the fetal membranes(PPROM), i.e., the chorioamnion and/or the occurrence of the risk ofpreterm labor (PTL). The inventive method provides greater accuracy thanthe currently available procedure using the detection of fFN.

More particularly, we have discovered that the excessive production ofthe reactive oxygen species (ROS), hypochlorous acid (HOCl), orexcessive decreases of antioxidant defenses lead to focal areas ofincreased collagen destruction in the chorioamnion and preterm prematurerupture of membranes (PPROM), or increased production of prostaglandinand preterm labor (PTL). Increased dietary intake of vitamins C and Etogether offers defense against HOCl-induced membrane damage andprostaglandin production and may prevent PPROM and PTL (32, 33).

The inventive method comprises determining the likelihood of theoccurrence of preterm premature rupture of membranes or increasedproduction of prostaglandin and preterm labor in a pregnant female byobtaining a sample of the females vaginal secretions, and analyzing thesecretions for the presence and amount of hypochlorous acid by measuringthe amount of 3-chlorotyrosine in the sample.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows the steps of producing an N-acetyl-3-chlorotyrosinecontaining antigen for subsequent antibody production.

FIG. 2 shows the steps of producing an N-acetyl-3-chlorotyrosine and anN-acetyl-3,5-dichlorotyrosine containing antigen for subsequent antibodyproduction.

FIG. 3A shows the first three steps of producing a novel3-chlorotyrosine containing hapten(3-(3-chloro-4-hydroxy-benzyl)-6-mercaptomethyl-piperazine-2,5-dione)which is ideal for coupling to a carrier protein to form an antigen forsubsequent antibody production.

FIG. 3B shows the next three steps of producing a novel 3-chlorotyrosinecontaining hapten(3-(3-chloro-4-hydroxy-benzyl)-6-mercaptomethyl-piperazine-2,5-dione)which is ideal for coupling to a carrier protein to form an antigen forsubsequent antibody production.

FIG. 3C shows the final steps of producing a novel 3-chlorotyrosinecontaining hapten(3-(3-chloro-4-hydroxy-benzyl)-6-mercaptomethyl-piperazine-2,5-dione)which is ideal for coupling to a carrier protein to form an antigen forsubsequent antibody production.

DETAILED DESCRIPTION OF THE INVENTION

The following description is provided to enable any person skilled inthe art to make and use the invention and sets forth the best modescontemplated by the inventor of carrying out his invention. Variousmodifications, however, will remain readily apparent to those skilled inthe art, since the general principles of the present invention have beendefined herein specifically to provide a method using 3-chlorotyrosinein vaginal secretions to diagnose an increased likelihood of pretermlabor.

Reactive oxygen species are tissue damaging molecules used by phagocytesto kill bacteria and which leak from the electron transport system ofthe mitochondria during cell respiration. They are characterized eitherby a single unpaired electron in the outer orbit (examples: superoxide,hydroxyl radical, and nitric oxide) or as molecules that share anelectron in their outer orbit (examples: hypochlorous acid, hydrogenperoxide). Their tissue damaging actions result in part as theyaggressively seek to extract an electron from an adjacent molecule torecreate electron stability. Hydrogen atoms in the double bonds or tailof polyunsaturated fats offer available targets for this form ofextraction, thereby setting the stage for lipid peroxidation as covalentbonds are disrupted. ROS also denature proteins, damage DNA, adverselyalter collagen and disrupt the integrity of cell membranes (10).

There are certain clinical states that are associated with PPROM knownto produce ROS or consume antioxidants. These include:

-   -   1. Infection: Chronic infection or inflammation most likely is        part of each PPROM case. In-vitro studies indicate that        metalloproteinase-producing bacteria such as Pseudomonas        aeruginosa, Staphylococcus aureus, and Bacteroides        melaninogenicus can decrease bursting load and work-to-rupture        in the fetal membrane. Studies also have shown that        Staphylococcus aureus and group B streptococci can decrease        chorioamnion tensile strength. In one study, when membranes were        exposed to activated neutrophils, similar findings were        documented which were augmented further in the presence of        Staphylococcus aureus (18, 25, 19).    -   2. Cigarette smoking: Smoking is a leading risk factor for        PPROM. Tobacco smoke contains a complex mixture of ROS that        induce systemic oxidative damage to multiple tissues. At the        level of the chorioamnion, cigarette smoking most likely        consumes antioxidants, thereby making the tissues more        vulnerable to the normal process of ROS generation (24).    -   3. Second trimester bleeding: Second trimester bleeding probably        increases the risks of PPROM by providing a fertile medium for        bacterial growth or by releasing iron as red cells degrade.        Iron, in turn, could catalyze formation of ROS.    -   4. Cocaine use: The relationship of cocaine use to PPROM is        believed to result from ischemia and then reperfusion caused by        the drug and which generates free radicals as reperfusion        follows the ischemic process (37).

ROS can degrade collagen in vitro. Hypochlorous acid, the principal ROSreleased by phagocytes, is considered the main ROS operative in thegeneration of PPROM and PTL. Hypochlorous acid damages collagen in thechorioamnion by blocking the tissue inhibitors of themetalloproteinases, thereby upregulating their activity to result incollagen degradation. Hypochlorous acid also attacks the proline and4-hydroxyproline sites that normally provide cross linkages in thecollagen helix as support systems (20).

Membrane segments, when incubated with varying doses of hypochlorousacid, demonstrate decreased collagen 1 staining and alterations in theamnion epithelium. Similar findings have been observed when chorioamnionsegments have been incubated with superoxide but since this is a muchweaker ROS, the tissue damage is less than that observed withhypochlorous acid (22, 12).

In other tissues, hypochlorous acid alters a range of otherwise normalcellular functions. When red blood cell membranes are exposed tohypochlorous acid, cell membrane fluidity and membrane Na⁺/K⁺/Mg⁺⁺ATPase activity are compromised (36).

Epithelial amnion cells also react in other ways to exposure to reactiveoxygen species. In vitro studies demonstrate that monolayers ofamniocytes, when exposed to the ROS exhibit increased intracellularcalcium, decreased intracellular magnesium, and release of arachidonicacid (precursor for prostaglandin production in the inflammatorypathway). When the increase in intracellular calcium is prevented,release of arachidonic acid is decreased (16). Amniocytes exposed totumor necrosis factor alpha and interleukin-1 also demonstratedincreased production of MMP, and prostaglandin E2 (26). These findingssuggest a possible relationship between PPROM and preterm labor.

The body has a number of antioxidant defenses, which are capable ofscavenging reactive oxygen species, and to minimize or preventROS-tissue induced damage. Enzyme antioxidants also scavange ROS.Superoxide dismutase (SOD) converts superoxide to hydrogen peroxide.Catalase or glutathione peroxidase converts superoxide to hydrogenperoxide or oxygen gas (O₂) and water, respectively. Other importantantioxidants like albumin, uric acid, and bilirubin bind trace metalsthereby preventing them from participating in free radical production.

Dietary antioxidants offer a different level of protection. Ascorbicacid (vitamin C), a water soluble vitamin, cannot be synthesized in thehuman body and is obtained by consuming fruits and vegetables such asred and yellow peppers, broccoli, strawberries and oranges. Vitamin E(tocopherol-OH) is a lipid soluble antioxidant and the most importantchain-breaking defense against lipid peroxidation. It is found in plantoils and nuts. Vitamin C and vitamin E now are believed to worksynergistically. Vitamin E, by donating a hydrogen atom, blocks theprogression of lipid peroxidation but becomes a free radical(tocopherol-O.). Ascorbic acid then donates a hydrogen atom to thetocopheryl radical, thereby recycling it back into the lipid interfaceas tocopherol-OH; in this process ascorbic acid becomes dehydroascorbicacid, a weak radical which is excreted in the urine. As long as adequatevitamin C is available, vitamin E is continually recycled (11).

In vitro evidence has shown that vitamin C and E can prevent ROS-induceddamage to the chorioamnion. In one series, membrane segments wereincubated for four hours in graded dose of hypochlorous acid. This levelof exposure produced damage to collagen 1 and alterations in amnionepithelium architecture. When these membranes were incubated first withvitamins C and E, rinsed, and then exposed to hypochlorous acid, noROS-induced damage was detected. This is the first evidence that vitaminC and vitamin E may offer protection to the chorioamnion fromROS-induced exposure (22).

Recent data indicate that vitamins C and E distribute through thematernal-fetal-amniotic fluid compartments differently. For thesestudies, plasma vitamin E concentrations were determined by reversedphase HPLC and standardized to cholesterol. Vitamin C was determined bythe 2,4-DNPH method. The results indicate that vitamin E is higher inthe maternal plasma than fetal plasma. For vitamin C, the distributionis different. Amniotic fluid contains the highest concentrations ofvitamin C with less being noted in fetal plasma and the lowestconcentrations in maternal plasma (34).

Low maternal plasma and leukocyte ascorbic acid concentrations (vitaminC) have been linked to PPROM. Specifically, plasma ascorbic acid levelsat six to eight months of pregnancy were lower in patients at termexperiencing PROM as compared with patients entering labor with intactmembranes (29).

Low leukocyte vitamin C levels at 20 weeks' gestation also wereassociated with an increase in PPROM (6). In amniotic fluid,significantly lower levels of ascorbic acid have been found in smokersthan nonsmokers (3). Studies of vitamin E concentrations and theirrelationship to PPROM have not been published.

Many investigators believe that women in the United States are nottaking adequate levels of vitamins C and E in their prenatal vitamins.Prenatal vitamins contain 60 to 100 mg of vitamin C and 10 to 30 IU ofvitamin E. The dosage of vitamin C was established initially to preventscurvy; the dosage of vitamin E was chosen which matched that taken inwith a normal diet. Subsets of the population are poorly supplementedwith dietary vitamin C and E. Data from the first NHANES (1971-1974)showed that African Americans had four-fold lower plasma concentrationsfor vitamin E and ten-fold lower plasma concentrations for vitamin Cthan Caucasians (Block 1988). If vitamins are to be used as therapy andnot simply to combat dietary deficiencies, then dosages of vitamins Cand E five to ten times those found in prenatal vitamins must bestudied.

Hypochlorous acid produced by neutrophils, is an initiating event in thecascade leading to release of arachidonic acid from amnion epithelialcells and the subsequent production of prostaglandins leading to PTL.Hypochlorous acid (HOCl) is a primary biomarker for neutrophil-derivedinflammation. HOCl is formed as myeloperoxidase, a major phagocyticprotein, catalyzes the 2-electron peroxidation of chloride. In fact,myeloperoxidase is the only human enzyme capable of producing HOCl atphysiologic concentrations of halide ions. Since halogenated moleculesare formed by such limited pathways, they serve as good markers forphagocytic-predicted tissue damage (14, 30).

We have discovered that the measurement of hypochlorous acid in vaginalsecretions of women at risk for preterm labor, provides a valuablebiomarker. Unfortunately, hypochlorous acid is not sufficiently stableto be detectable at a distant site. 3-chlorotyrosine, however, is astable oxidized product generated from hypochlorous acid and inaccordance with the present invention, it can be detected and measuredin the vaginal secretions of women at risk for PTL and provide anaccurate determination of the presence and amount of HOCl in thesecretions. This, in turn, provides an accurate indicator of thelikelihood of PTL and/or PPROM so that therapeutic measures can betaken.

Because of its unique pathway for formation, 3-chlorotyrosine detectedin vaginal secretions reflects the release of HOCl further up in thepregnant uterus. 3-chlorotyrosine has been detected in elevated levelsin patients with coronary artery disease when low density lipoprotein(LDL) is exposed to HOCl but not when LDL is exposed to other ROS suchas OH., copper, iron, heme, glucose, peroxynitrite, horseradishperoxidase, lactoperoxidase or lipoxyglucose. It appears that3-chlorotyrosine is a stable marker of LDL oxidation by HOCl. Moreover,it is increased markedly in atherosclerotic tissue obtained at vascularsurgery and is mildly increased in plasma LDL of older men with coronaryartery disease but not healthy young men (13).

The presence and level of the 3-chlorotyrosine in the sample of vaginalsecretion can readily be determined by immunoassay utilizing eitherpolyclonal or monoclonal antibodies to the 3-chlorotyrosine modifiedproteins. Radioimmunoassays and enzyme immunoassays, e.g., anenzyme-linked immunosorbent assay (ELISA)) known in the art, see forexample (9).

In carrying out the procedure, a capture ELISA or a sandwich ELISA isused for the detection and quantitative analysis of 3-chlorotyrosine ina clinical sample. This involves as a first step, raising antibodiesagainst 3-chlorotyrosine coupled to a carrier such as bovine serumalbumin (BSA) because 3-chlorotyrosine itself is a hapten. As shown inFIG. 1, most standard procedures for coupling the hapten to proteinrequire first protecting the amino group of 3-chlorotyrosine—for exampleby acetylation. This may be carried out by chemical means, e.g.,Schotten-Baumann reaction. Those of ordinary skill in the art willrecognize that a variety of other amino protective reagents can beemployed. N-acetyl-3-chlorotyrosine (N-acetyl-3-CIY) is then coupled tothe BSA using any of a number of well-know linking reagents. The drawingshows coupling though the carboxyl group of the modified tyrosine, whichcoupling can be carried out, for example, by use of carbodiimidereagents, as is well known to those of ordinary skill in the art. FIG. 2shows an alternative approach in which treatment of N-acetyl-tyrosinewith hypochlorous acid produces both N-acetyl-3-chlorotyrosine andN-acetyl-3,5-dichlorotyrosine both of which are subsequently coupled toa carrier protein. Both the monochloro and dichloro product are found invaginal secretions although the former is much more abundant. By usingboth compounds to form antigens, the resulting antibodies show enhancedsensitivity while remaining highly selective.

FIG. 3 (FIGS. 3A, 3B, and 3C) shows the synthesis of a novel hapten(3-(3-chloro-4-hydroxy-benzyl)-6-mercaptomethyl-piperazine-2,5-dione)which is optimized for sulfhydryl linking to a carrier protein toproduce an effective neoantigen for raising antibodies to3-chlorotyrosine. The illustrated synthesis combined with linkage to aprotein modified with such well-known N-hydroxysuccinimide-maleimidecontaining linkers as SMCC (4-(Maleimidomethyl)cyclohexanecarboxylicAcid N-Hydroxysuccinimide Ester) allows simple creation of an antigenhaving 3-chlorotyrosine epitopes. Note that in the figures the carrierprotein is indicated at BSA (bovine serum albumin) but other suitablecarrier proteins such as thyroglobulin and keyhole limpet hemocyanin canbe used instead.

According to FIG. 3A in a first synthetic step chlorine gas is bubbledinto an ice-cold methanol solution of tyrosine methyl ester (see, Y.Terada, Tetrahedron Lett. 1996, 37(48):8791-8794).

In a second step benzyl choloroformate and sodium hydroxide solutionwere added simultaneously to a solution of L-cysteine in sodiumhydroxide to yield N,S-dicarbobenzoxy-L-cysteine at 90% yield (see, A.Berger, J. Niguchi, E. Katchalski, J. Am. Chem. Soc. 1956,78:4483-4487).

In a third step the addition product, N,S-dicarbobenzoxy-L-cysteine, andthe 3-chlorotyrosine methyl ester are reacted for twelve hours withdicyclohexylcarbodiimide in N,N-dimethyl-formamide (see, K. Jost, J.Rudinger, F. Sorm, Coll. Czech. Chem. Commun. 1961, 26:2496-2510) togive a 90% yield of a dipeptideN,S-dicarbobenzoxy-L-cysteinyl-L-3-chlorotyrosine methyl ester.

The dipeptide (see FIG. 3B) is then treated in a fourth step with sodiummethoxide in methanol (see, L. Zervas, I. Photaki, N. Ghelis, J. Am.Chem. Soc. 1963, 85:1337-1341) for 10 min. and in a fifth step thereaction mixture was acidified with acetic acid and titrated with iodinein methanol to yield the dimerN,N′-biscarbobenzoxy-L-cystinyldi(3-chloro)tyrosine dimethyl ester at90% yield (see, L. Zervas, I. Photaki, N. Ghelis, J. Am. Chem. Soc.1963, 85:1337-1341).

The dimer is then treated in a sixth step for 15 min. with 30%hydrobromic acid in acetic acid solution (see, K. Blaha. Coll. Czech.Chem Commum. 1969, 34:4000-4005) to give an 89% yield ofL-cystinyldi(3-chloro)tyrosine dimethyl ester.

The L-cystinyldi(3-chloro)tyrosine dimethyl ester is then treated in aseventh step for eight hours with ammonia in methanol (see, E. Fischer,Chem Ber. 1906, 39:2893) to give a 60% yield. This product (see FIG. 3C)is then treated in an eighth step with tri-n-butylphosphine in aqueousmethanol to cleave the cystine-disulfide bond (see, S. A. Khan, B. W.Erickson, J. Am. Chem Soc. 1981, 103:7373-7376) to give a 78% yield ofthe final product,3-(3-chloro-4-hydroxy-benzyl)-6-mercaptomethyl-piperazine-2,5-dione(overall yield of about 30%).

These antigens are advantageous because a single (or dual) neoantigen isused which allows a high degree of selectivity for the detection ofprotein-bound 3-chlorotyrosine. In the prior art some use has been madeof hypochlorous acid exposed proteins as antigens. This provides lessselective antibodies because such proteins are likely to contain othermodified amino acids including modified lysine, cysteine, and histidineresidues are obtained.

Using the antigens described above, the antibodies are raised forexample in a laboratory animal such as rabbit. This can be carried outby injecting the antigen followed by a booster inoculation with the sameprotein (usually, at least four weeks after priming) and collectingantisera from the animal after an additional period of four weeks.Alternatively, these antigens can be used to raise monoclonal antibodiesusing methods well known to those of ordinary skill in the art.Subsequently, the antisera may optionally fractionated usingchromatographic and similar techniques to yield purified IgG.

A fraction of the antibody obtained. (e.g., the rabbit antisera dilutedto 2 μg per microtiter well in phosphate buffered saline (PBS)) is thenbound to standard ELISA plates by overnight incubation at 4° C. Theplates are washed with PBS containing a surfactant such as 0.05% Tween20 (polysorbate 20). The clinical sample to be analyzed is appropriatelydiluted, and preferably serially diluted samples, (10-100 μl/well) areadded to ELISA plates (about 100 μl/well)

After 1 hour incubation at 37 ° C., the plates are washed 3-4 times.Then a suitable amount of a secondary antibody (depending on the sourceof the primary antibody—e.g., if the primary antibody is raised inrabbits, then goat-antirabbit antibody is used as the secondaryantibody) conjugated to a fluorescent or chemiluminescent label or anenzyme such as alkaline phosphatase or peroxidase is added. Thesecondary antibody can be monoclonal or a polyclonal antibody.

After washing the plate with PBS, about 50 μl of an appropriate reactionor substrate solution (in the case of a chemiluminescent or an enzymelabel) is added per well. After a 1 hour reaction period, the absorbanceat a suitable wavelength (e.g., 450-590 nm) is measured using a platereader or by optical density scanning of the plate. Fluorescent labelscan be read directly by a fluorescence plate reader.

The ability to measure 3-chlorotyrosine, a stable biomarker ofhypochlorous acid in vaginal secretions of at-risk women for pretermlabor, significantly increases our ability to detect these at-risk womenand implement labor-inhibiting medications including antioxidantcompounds intended to block the action of hypochlorous acid. Treatmentcan be instituted only when actually needed. Further, by monitoring the3-chlorotyrosine it is possible to see if the treatments are having apositive effect. Various other immunoassays and detection techniqueswill be apparent to those skilled in the art after reading the presentdisclosure without departing from the spirit and scope of the invention.For example, it is known to utilize mass spectrometry to measure3-chlorotyrosine in human tissue with attomole sensitivity (14). It isintended that all such other techniques for measuring 3-chlorotyrosinein vaginal secretions be included within the scope of the presentinvention.

The following claims are thus to be understood to include what isspecifically illustrated and described above, what is conceptuallyequivalent, what can be obviously substituted and also what essentiallyincorporates the essential idea of the invention. Those skilled in theart will appreciate that various adaptations and modifications of thejust-described preferred embodiment can be configured without departingfrom the scope of the invention. The illustrated embodiment has been setforth only for the purposes of example and that should not be taken aslimiting the invention. Therefore, it is to be understood that, withinthe scope of the appended claims, the invention may be practiced otherthan as specifically described herein.

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1. A method for determining the presence of hypochlorous acid in vaginalsecretions comprising measuring the presence and amount of3-chlorotyrosine in the vaginal secretions.
 2. A method for determiningthe likelihood of preterm premature rupture of fetal membranes orpreterm labor in a pregnant female comprising the steps of: obtaining asample of vaginal secretions from the female; and analyzing the samplefor the presence and amount of hypochlorous acid by measuring the amountof 3-chlorotyrosine in the sample.
 3. A method for therapeuticallytreating a pregnant female to minimize the likelihood of pretermpremature rupture of fetal membranes or preterm labor comprising thesteps of: obtaining a sample of vaginal secretions from the female;measuring the presence and amount of 3-chlorotyrosine in the vaginalsecretions wherein an increased amount of 3-chlorotyrosine represents anincreased likelihood of preterm premature rupture of fetal membranes orpreterm labor; and administering an amount of dietary antioxidant to thefemale if the likelihood is increased.
 4. The method of claim 3, whereinthe dietary antioxidant is selected from the group consisting of vitaminC and vitamin E.
 5. A method for determining the presence ofhypochlorous acid in female vaginal secretions comprising measuring thepresence and amount of 3-chlorotyrosine in the vaginal fluid using anELISA assay.
 6. A novel hapten for raising antibodies to3-chlorotyrosine, said hapten comprising3-(3-chloro-4-hydroxy-benzyl)-6-mercaptomethyl-piperazine-2,5-dione. 7.A neoantigen for raising antibodies to 3-chlorotyrosine comprising acarrier protein bound to the hapten of claim 6 by way of a covalentlinkage.
 8. The neoantigen of claim 7, wherein the carrier protein isselected from the group consisting of bovine serum albumin, keyholelimpet hemocyanin and thyroglobulin.
 9. The neoantigen of claim 7,wherein the covalent linkage includes a sulfur atom.
 10. A method forraising antibodies to 3-chlorotyrosine comprising the use of an antigenformed by covalently linking3-(3-chloro-4-hydroxy-benzyl)-6-mercaptomethyl-piperazine-2,5-dione to acarrier protein.
 11. The method of claim 10, wherein the carrier proteinis selected from the group consisting of bovine serum albumin, keyholelimpet hemocyanin and thyroglobulin.
 12. A method for raising antibodiesto 3-chlorotyrosine comprising using an antigen formed by covalentlylinking N-acetyl-3-chlorotyrosine to a carrier protein.
 13. The methodof claim 12, further comprising using an antigen formed by covalentlylinking N-acetyl-3,5-dichlorotyprosine to a carrier protein.
 14. Themethod of claim 12, wherein the carrier protein is selected from thegroup consisting of bovine serum albumin, keyhole limpet hemocyanin andthyroglobulin.